This invention relates to novel betulinic acid derivatives and a composition containing betulinic acid derivatives, processes for preparation of such betulinic acid derivatives and method of treatment of tumor and such proliferative disease using these derivatives. This invention also relates to the use of the novel betulinic acid derivatives for inhibiting and/or preventing tumor associated angiogenesis, more specifically angiogenesis associated with prostate, lung, ovary and colon cancers.
Betulinic acid is a pentacyclic triterpene. It can derived from several natural (botanical) sources. It can also be chemically derived from betulin, a substance found in abundance in the outer bark of white birch trees (Betula alba). Betulinic acid has been found to selectively kill human melanoma cells (Nature Medicine, Vol.1(10),1995, WO 96/29068). The cytotoxic potential of betulinic acid was tested using three human melanoma cell lines, Mel-1, Mel-2, and Mel-4. The growth of all the cell lines was inhibited significantly by treatment with betulinic acid. The effectiveness of betulinic acid against melanoma cancer cells was also tested using athymic mice. It seems to work by inducing apoptosis in cancer cells.
The anti-cancer activity of betulinic acid and some of its derivatives has also been demonstrated using mouse sarcoma 180 cells implanted s.c. in nude mouse (JP 87,301,580), inhibition of growth of p388 lymphocytic leukemia cells in vitro (Choi.Y-H et al., Planta Medica Vol.XLVII,511-513,1988) and inhibiting growth of cancer cells, particularly by inhibiting ornithine decarboxylase (Yasukawa, K et al, Oncology 48:72-76,1991; WO 95/04526).
Recently, the applicants reported anti-leukemia and anti-lymphoma activity and anti-prostate, anti-lung and anti-ovarian cancer activity of betulinic acid and its derivatives with ED50 values less than 4.0 xcexcg/ml. (U.S. application Ser. No. 09/040,856 filed on Mar. 18, 1998 and U.S. application Ser. No. 09/251,309 filed on Feb. 17, 1999). Further, antiangiogenic activity of betulinic acid and its derivatives was recently reported by the applicants in U.S. application Ser No. 09/166,809 filed on Oct. 06, 1998 wherein betulinic acid and its derivatives were shown to inhibit the formation of tube-like-structures (TLS) of endothelial cells when grown on Matrigel coated surface. The endothelial cell anti-proliferative activity along with anti-TLS activity was shown to suggest the anti-angiogenic activity of betulinic acid derivatives.
Anderson et al (WO 95/04526) disclose that for certain cancers to spread throughout a patients"" body, a process termed metastasis, cell-cell adhesion must take place. Specifically, cancer cells must migrate from their site of origin and gain access to blood vessel to facilitate colonization at distant sites. Certain cancer cells are known to adhere to E-Selectin via E-Selectin ligands on their cell surface and this event is one component of the metastasis process. Betulinic acid and its derivatives interfere with Selectin binding. Betulinic acid inhibited P-Selectin binding to 2,3, sLex, a chemical known: to bind to P-Selectin, with an IC50 of 125 uM. Also it inhibited P-Selectin binding HL-60 cells in a dose-dependent way with an IC50 of 0.75 mM. Betulinic acid and derivatives also significantly interfere with the binding to colon cancer cells, LS174T to E-Selectin.
Dasgupta et al (WO 96/29068) disclosed a method and composition for inhibiting tumor growth using the active compound betulinic acid. The invention provides a method and composition for inhibiting tumor growth and, particularly, for inhibiting growth of melanoma using a natural product derived compound. The invention also provides a treatment method using betulinic acid to prevent growth or spread of cancer cells, wherein betulinic acid is applied in a topical preparation.
Pezzuto et al (U.S. Pat. No. 5,869,535) disclose method and composition for probes inhibiting tumor growth using betulinic acid or a derivative thereof. Betulinic acid has been isolated from stem bark of Ziziphzus mauritiana, by mediating a selective cytotoxic profile against human melanoma in a subject panel of human cancer cell lines, conducting a bioassay directed fractionation based on the profile of bioactivity using cultured human melanoma cells (MEL-2) as the monitor, and betulinic acid has been obtained therefrom as the active compound. The resulting betulinic acid can be used to inhibit tumor growth or can be converted to a derivative to prevent which prevents or inhibits tumor growth. The invention also provides a treatment method using betulinic acid to present the growth or spread of cancerous cells, wherein betulinic acid or derivatives thereof is applied in a topical preparation. Betulinic acid was found to inhibit in vitro growth of MEL-2 cells. However, none of the other cell lines tested [A431 (squamous cells), BC-1 (breast), COL-2 (colon), HT1080 (sarcoma), KB (human oral epidermoid carcinoma), LNCaP (prostate), LU-1 (lung), U373 (glioma) and ZR-75-1 (breast)] were affected by betulinic acid (ie., ED50 values of greater than 20 xcexcg/ml).
Lee et al (WO 96/39033) disclose betulinic acid and dihydrobetulinic acid acyl derivative to have potent anti-HIV activity. The C3-hydroxy, C17-carboxylic acid and C20-exomethylene groups have been modified. Anti-HIV assays indicate potent anti-HIV activity of betulinic acid and dihydrobetulinic acid derivatives in acutely infected H9 lymphocytes with EC50 values of less than 1.7xc3x9710xe2x88x925 xcexcM respectively.
The invention provides a method of treating angiogenesis by administering a pharmaceutically effective dosage of betulinic acid derivatives. This invention also provides for novel betulinic acid derivatives and compositions containing them with pharmaceutically acceptable additives, diluents, carriers and excipients with or without betulinic acid.
Another object of the invention relates to providing novel betulinic acid derivatives, which are used for inhibiting angiogenesis.
Another object of the invention is to provide a compound and compositions for treating, inhibiting and/or preventing angiogenesis using a natural product-derived compound and its derivatives.
Another object of the invention is to provide a treatment method using betulinic acid derivatives to inhibit angiogenesis, wherein the derivatives are administered systemically.
Yet another object of the invention is to overcome the problem of high toxicity associated with standard antiangiogenic chemotherapeutic agents by using a natural product-derived compound, e.g., betulinic acid or its derivatives.
Still another object of the invention is to overcome the problem of insufficient availability associated with synthetic antiangiogenic anticancer agents by using readily available semisynthetic derivatives of betulinic acid.
Another object of the invention is to overcome the problem of high costs of synthetic antiangiogenic agents by utilizing the readily available natural product derived compound. e.g. betulinic acid and its derivatives which is expected to be less expensive than other chemotherapeutic drugs.
These and other objects of the present invention will become apparent from the description of the invention disclosed below, which descriptions are intended to limit neither the spirit or scope of the invention but are only offered as illustrations of the preferred embodiments of the invention.
The above objects and others have been achieved by providing novel betulinic acid derivatives of formulae 1 and 2 which are described in the present description.
The present invention provides a pharmaceutical composition useful for preventing/inhibiting angiogenesis. Betulinic acid derivatives inhibit endothelial cell proliferation and exhibiting high endothelial cell specificity thereby specifically targeting endothelial cells. The derivatives also inhibit the formation of tube-like-structures (TLS) of endothelial cells when grown on Matrigel coated surface. The endothelial cell anti-proliferative activity along with anti-TLS activity very strongly suggests the anti-angiogenic activity of betulinic acid derivatives.
The method comprises administering a therapeutically effective dose betulinic acid derivatives other alone or in a pharmaceutical composition containing the compounds so as to kill, inhibit or prevent the multiplication of tumor associated endothelial cells. In a preferred embodiment, pharmaceutically acceptable carriers, diluents, excipients and/or solvents are used with betulinic acid/or its derivatives. The method of treatment of the present invention may be particularly useful in inhibiting angiogenesis.
The novel derivatives of betulinic acid have a basic skeleton of betulinic acid as shown herebelow in Figure 1. 
wherein R, R1 R2, R3, R4, R5, R6 and R7 independently or in combination represent the following groups:
R is H;
R1 is H, Br, Cl, F or I;
R2 is H and R3 is OH, OCO(CH2)nCH3 (where n=0 to 14), OCOC(CH3)3, OCO(CH2)nX (where n=1 to 7, X=H, Cl, Br, F), OCOCH2C6HnX [n=2 to 4, X=H, Cl, Br, F, I, CN, NO2, NH2, CF3, CHCl2, OH, OCH3, OC2H5, CHCl2 or CnH2n+1(n=1 to 7)], OSO2(CH2)nX (where n=1 to 7, X=H or Cl), OSO2ONH2, OCOC6HnX [n=0 to 4, X=H, Cl, Br, F, I, CN, NO2, NH2, CF3, OH, OCH3, OC2H5, CHCl2 or CnH2n+1(n=1 to 7)], NH2, NH(CH2)nOR [(n=2 to 4), R=H or COCH3], NHR, N(R)2 [where R=CH3, C2H5, C3H7, C4H9], NHC6HnX, NHCH2C6HnX (where n=2 to 4), NHCH2C10HnX (n=2 to 7) [X=H,Cl, Br, F, I, CHCl2, CN, CF3, CHCl2, OH, OCH3, OC2H5 or CnH2n+1(n=1 to 7)], RCH2NOH (R=H,CH3,C2H5,C3H7,C4H9), NHOR (R=H, COCH3, COC6HnX, OCH2C6HnX, OC6HnX) [n=2 to 4, X=Cl, Br, F, I, CF3, CHCl2, CN, NO2, CH3, NH2, OH, OCH3, OC2H5 or CnH2n+1(n=1 to 7)], N=CHC6HnX (where n=2 to 4), N=CHC10HnX (n=2 to 6)[X=H, Cl, Br, F, I, CF3, CN, NO2, NH2, OH, OCH3, OC2H5 or CnH2n+1 (n=1 to 3)], OCO(CH2)nNH2 (n=2 to 8), NHCO(CH2)nX (X=H,Cl or Br, n=1 to 4), NHCOC6HnX, NHCOC10HnX (n=2 to 6), NHCOCH2C6HnX (n=2 to 4), NHCOCH2C10HnX (n=2 to 6)[X=Cl, Br, F, I, CF3, CN, NO2, NH2, OH, OCH3, OC2H5, CHCl2 or CnH2n+1(n=1 to 7)], NHCOC6H4COOH, NHCOC6Hn(COOH)X [where n=2 or 3, X=H, Cl, Br, F, NO2 or NH2), OCOC6H4COOH, OCOC6Hn(COOH)X (where n=2 or 3, X=H, Cl, Br, F, NO2 or NH2), OCOCHRR1, (R=H, CH3 or Ph; R1=OH, Cl, Br or OCOCH3), NHNHC6HnX (n=2 to 4), NHNHCH(OH)C6HnX (n=2 to 4), NHNHC10HnX (n=2 to 6), NHNHCH(OH)C10HnX (n=2 to 6)[X=Cl, Br, F, I, OH, OCH3, OC2H5, NO2, NH2, CHCl2, CF3 or CnH2n+1(n=1 to 7)], OCOCHxe2x95x90C(R)2 (R is H, CH3 or C2H5), Oxe2x80x94COxe2x80x94CHxe2x95x90CHxe2x80x94COOH, Oxe2x80x94COxe2x80x94C(Br)xe2x95x90CHCOOH, OCOCH2C(R)2COOH (R=H or CH3), OCO(CH2)nCOOH (n=0 to 3), 
[R=NH2, NHC6HnX (n=2 to 4), NHC10HnX (n=2 to 6), NHCO(CH2)nX (n=1 to 16)[X=H, Cl, F, Br], NHCOC6HnX, NHCOCH2C6HnX (n=2 to 4), NHCOC10HnX (n=2 to 6), Nxe2x95x90CHC6HnX (n=2 to 4), Nxe2x95x90CHC10HnX (n=2 to 6), NHCH2C6HnX (n=2 to 4), NHCH2C10HnX (n=2 to 6)[X=H, Cl, Br, F, I, CN, NO2, NH2, CF3, CHCl2, OCH3, OC2H5 or CnH2n+1 (n=1 to 7), NHSO2(CH2)nX (n=1 to 7), NHSO2C6HnX (n=2 to 4)[X=H, Cl, Br, F, CH3, NO2 or NH2],
R2 and R3 together are O, NNHC6HnX, NNHCOC6HnX (n=2 to 4), NNHC10HnX (n=2 to 6), NNHCOC6HnX (n=2 to 6), NC6HnX (n=2 to 4), NC10HnX (n=2 to 6), [X=H, Cl, Br, F, I, CN, NO, NO2, NH2, CF3, CHCl2, OH, OCH3, OC2H5 or CnH2n+1 (n=1 to 7)], NNHC6HnBrX [(n=2 or 3), X=F, Cl, NO2, NH2, OCH3, OC2H5, CnH2n+1 (n=1 to 7)], NOSO3H, Nxe2x80x94OX, NHOX [X being H, CH3, C2H5, COCH3, SO2C6H4CH3, COC6HnX, C6HnX, CH2C6HnX [(n=2 to 4) X=H, Cl, Br, F, I, CN, NO2, NH2, OH, OCH3, OC2H5, CnH2n+1 (n=1 to 7]. CF3 or CHCl2], NNHR [R is CH3, C2H5, C2H4OY, Y=H, alkyl, phlenyl, benzyl or its substituted derivative with Cl, Br, F, I, NO2, NH2, CF3, CHCl2, OH, OCH3, OC2H5 or CnH2n+1 (n=1 to 7)],
R7 is O and R4 is H, OH, Cl, N3, NH2, OR (R=CH3, C2H5, C3H7, C4H9), O(CH2)nCOY (n=1 to 3)[Y=OH, OCH3, OC2H5, Cl, CN, N3, NH2], OCH2CH2OY [Y=H, CH3, C2H5, COCH3], OCOCHxe2x95x90C(R)2 (R=H, CH3 or C2H5), OCO(CH2)nX (n=1 to 16), (X=H, Cl, F or Br), OCOC6HnX (n=0 to 4), OCOCH2C6HnX (n=2 to 4)[X=H, Cl, Br, F, I, CN, NO2, CF3, CHCl2, OH, OCH3, OC2H5 or CnH2n+1 (n=1 to 7)], NH(CH2)nCH3 (n=0 to 9), NH(CH2)nCOOH (n=1 to 8), OCH2CHO, OCH2CHxe2x95x90NOX, OCH2CH2NHOX[X=H, CH3, SO2C6H4CH3, OCOCH3, OCOC6H5, phenyl or benzyl substituted derivatives], OCH2CHxe2x95x90NNHC6HnX, OCH2CH2NHNHC6HnX (n=2 to 4), OCH2CHxe2x95x90NNHC10HnX (n=2 to 6), OCH2CH2CH2NHNHC10HnX [X=H, Cl, Br, F, I, CN, CF3, CHCl2, NO2, NH2, OH, OCH3, OC2H5 or CnH2n+1 (n=1 to 7)], OCH2CH2N(R)2 (R=H, CH3, C2H5, C3H7, C4H9, C6H5, C6H5CH2 or its substituted derivative e.g.: Cl, Br, CN, F, I, NO2, NH2, CF3, CHCl2, OH, OCH3, OC2H5 or CnH2+1 (n=1 to 7)],
R4 is H and R7 is NOH, NHOR, Nxe2x80x94OR [R is H, CH3, C2H5, SO2C6H4CH3, COCH3, CH2C6HnX, COC6HnX (n=2 to 4), X=Cl, Br, F, I, CN, NO2, NH2, OH, OCH3, OC2H5, CF3, CHCl2 or CnH2n+1 (n=1 to 7)], RCH2NOH (R=H, CH3 or C2H5), NH2, NHSO2(CH2)nX (n=1 to 7), NHSO2C6HnX (n=2 to 5)[X=H, Cl, Br, CH3, NO2 or NH2], (NR)2 (R is H, CH3, C2H5, C3H7, C4H9, Phenyl or Benzyl or its substituted derivative), Nxe2x95x90CHC6HnX, NHCH2C6HnX (n=2 to 4), Nxe2x95x90CHC10HnX, NHCH2C10Hn[X (n=2 to 6) X=H, Cl, Br, F, I, CN, NO2, NH2, CF3, CHCl2, OH, OCH3, OC2H5 or CnH2n+1 (n=1 to 7)], NNHC6HnX, NHNHC6HnX, NHNHCH(OH)C6HnX, NNHCOC6HnX (n=2 to 4), NNHC10HnX, NNHCOC10HnX, NHNHC10HnX, NHNHCH(OH)C10HnX [where n=2 to 6, X=H, Cl, Br, F, I, CN, NO2, NH2, OH, OCH3, OC2H5, CnH2n+1 (n=1 to 7)], NHCOR [R is CH3, CH2Cl, CHCl2, CCl3, C2H5, C2H4Cl, C3H7, C3H6OH, C3H6Cl, C6H5, C6HnX, CH2C6HnX, COCH2C6HnX (n=2 to 4), C10HnX, CH2C10HnX, COCH2C10HnX (n=2 to 6), X=Cl, Br, CN, F, I, NO2, NH2, CF3, OH, OCH3, OC2H5, CHCl2 or CnH2n+1 (n=1 to 7)],
R5 is H or Br, R6 is CH3, CH2Br, CH2OR [R is CO(CH2)nX, (n=1 to 7: X=H, Cl, Br or F), CHO, CHNOY, CH2NHOY, [Y=H, CH3, C2H5, SO2C6H5, SO2C6H4CH3, CH2C6HnX, C6HnX (n=2 to 4), X=H, Cl, Br, F, I, CN, NO2, NH2, CF3, CHCl2, OH, OCH3, OC2H5, CnH2n+1 (n=1 to 7)], RCH2NOH [where R is H, CH3, C2H5, C3H7, C4H9], CH2NH2, CH2NHR or CH2N(R)2 [R is CH3, C2H5, C3H7, C4H9, C6H5, C6HnX or CH2C6HnX, COCH2C6HnX (n=2 to 4), CH2C10HnX, COCH2C10HnX (n=2 to 6)[X=H, Cl, Br, F, CN, I, NO2, NH2, OH, OCH3, OC2H5, CF3, CHCl2 or CnH2n+1 (n=1 to 7)], COOH, COCl, CONHR (R is alkyl or aryl substituted group), COxe2x80x94OCOR (R is alkyl or aryl substituted group), COCH2COR (R is OH, OCH3, OC2H5, NH2 or Cl), COCH2CH2OR [R is H, CO(CH2)nX (n=1 to 16), COC6HnX, COCH2C6HnX, (n=2 to 4, X=H, Cl, Br, CN, F, I, NO2, NH2, CF3, CHCl2, OH, OCH3, OC2H5 or CnH2n+1 (n=1 to 7)], COO(CH2)nH (n=1 to 5), COO(CH2)nCOY (n=1 to 5, Y=OH, OCH3, OC2H5, Cl or Br), CHxe2x95x90NC6HnX (n=2 to 4), CHxe2x95x90NC10HnX (n=2 to 6), CHxe2x95x90NNHC6HnX, CHxe2x95x90NNHCOC6HnX (n=2 to 4), CHxe2x95x90NNHC10HnX, CHxe2x95x90NNHCOC10HNX (n=2 to 6), CH2NHNHC6HnX (n=2 to 4), CH2NHNHC10HnX (n=2 to 6), CH2NHNHCH(OH)C6HnX (n=2 to 4), CH2NHNHCH(OH)C10HnX (n=2 to 6) [where X=H, Cl, Br, F, I, CN, CF3, NO2, NH2, CHCl2, OH, OCH3, OC2H5 or CnH2n+1 (n=1 to 7)],
R5 and R6 together is O, OH, O(CH2)nX (n=1 to 6, X=H, Cl or Br), OCOC6HnX, OCOCH2C6HnX [n=2 to 5, X=Cl, Br, F, I, CN, NO2, NH2, CF3, OH, OCH3, OC2H5 or CnH2n+1 (n=1 to 7)], O(CH2)nCOOH (n=1 to 3), NOR, NHOR (R=H, CH3, C2H5, C3H7, COCH3, COC6H5, phenyl or benzyl substituted derivatives), NH2, (NR)2 (R=H, CH3, C2H5, C3H7, C4H9, C6HnX, CH2C6HnX; n=2 to 5, X=Cl, Br, F, I, CF3, CN, NO2, NH2, OH, OCH3, OC2H5, CnH2n+1 (n=1 to 7)], NHCO(CH2)nX [n=1 to 16, X=Cl or Br], NHCOC6HnX, NHCOCH2C6HnX (n=2 to 4), NHCOC10HnX, NHCOCH2C10OHnX (n=2 to 6) (X=Cl, Br, F, I, CN, CF3, NO2, NH2, OH, OCH3, OC2H5, CnH2n+1 (n=1 to 7)], Nxe2x95x90CHC6HnX (n=2 to 4), Nxe2x95x90CHC10HnX (n=2 to 6), NHCH2C6HnX (n=2 to 5), NHCH2C10HnX (n=2 to 6), NNHC6HnX, NC6HnX, NHC6HnX (n=2 to 4), NC10HnX, NHC10HnX, NNHC10HnX (n=2 to 6), NNHCOC6HnX (n=2 to 4), NNHCOC10HnX (n=2 to 6), NR [R=C6HnX (n=2 to 5), C10HnX (n=2 to 7)[X=H, Cl, Br, Cl, F, I, CN, NO2, NH2, CF3, CHCl2, OCH3, OC2H5, OH or CnH2n+1 (n=1 to 7)].
Preparation of Betulinic Acid Derivatives.